Gene therapy for lysosomal storage diseases

ABSTRACT

This disclosure provides methods and compositions for treating lysosomal storage diseases in a subject. In one aspect of the invention, a transgene product is delivered to a subject by administering a recombinant neurotrophic viral vector containing the transgene to the brain. The viral vector delivers the transgene to a region of the brain which is susceptible to infection by the virus and which expresses the encoded recombinant viral gene product. Also provided are compositions for delivery of a transgene product to a subject by administering a recombinant neurotrophic viral vector containing the transgene to the subject&#39;s brain. The transgene product may be any that is deficient in a lysosomal storage disease.

FIELD OF THE INVENTION

This invention is related to the area of lysosomal storage diseases.

SUMMARY OF THE INVENTION

Gene therapy is an emerging treatment modality for disorders affectingthe central nervous system (CNS). CNS gene therapy has been facilitatedby the development of viral vectors capable of effectively infectingpost-mitotic neurons. The central nervous system is made up of thespinal cord and the brain. The spinal cord conducts sensory informationfrom the peripheral nervous system to the brain and conducts motorinformation from the brain to various effectors. For a review of viralvectors for gene delivery to the central nervous system, see Davidson etal. (2003) Nature Rev. 4:353-364.

Adeno-associated virus (AAV) vectors are considered useful for CNS genetherapy because they have a favorable toxicity and immunogenicityprofile, are able to transduce neuronal cells, and are able to mediatelong-term expression in the CNS (Kaplitt et al. (1994) Nat. Genet.8:148-154; Bartlett et al. (1998) Hum. Gene Ther. 9:1181-1186; andPassini et al. (2002) J. Neurosci. 22:6437-6446).

One useful property of AAV vectors lies in the ability of some AAVvectors to undergo retrograde and/or anterograde transport in neuronalcells. Neurons in one brain region are interconnected by axons to distalbrain regions thereby providing a transport system for vector delivery.For example, an AAV vector may be administered at or near the axonterminals of neurons. The neurons internalize the AAV vector andtransport it in a retrograde manner along the axon to the cell body.Similar properties of adenovirus, HSV, and pseudo-rabies virus have beenshown to deliver genes to distal structures within the brain (Soudas etal. (2001) FASEB J. 15:2283-2285; Breakefield et al. (1991) New Biol.3:203-218; and deFalco et al. (2001) Science, 291:2608-2613).

Several groups have reported that the transduction of the brain by AAVserotype 2 (AAV2) is limited to the intracranial injection site (Kaplittet al. (1994) Nat. Genet. 8:148-154; Passini et al. (2002) J. Neurosci.22:6437-6446; and Chamberlin et al. (1998) Brain Res. 793:169-175).Recent reports suggest that retrograde axonal transport of neurotrophicviral vectors can also occur in select circuits of the normal rat brain(Kaspar et al. (2002) Mol. Ther. 5:50-56 (AAV vector); Kasper et al.(2003) Science 301:839-842 (lentiviral vector) and Azzouz et al. (2004)Nature 429:413-417 (lentiviral vector). Roaul et al. (2005) Nat. Med.11(4):423-428 and Ralph et al. (2005) Nat. Med. 11(4):429-433 reportthat intramuscular injection of lentivirus expressing silencing humanCu/Zn supreoxide dismutase (SOD1) interfering RNA retarded disease onsetof amyotrophic lateral sclerosis (ALS) in a therapeutically relevantrodent model of ALS.

Cells transduced by AAV vectors may express a therapeutic transgeneproduct, such as an enzyme to mediate beneficial effectsintracellularly. These cells may also secrete the therapeutic transgeneproduct, which may be subsequently taken up by distal cells where it maymediate its beneficial effects. This process has been described ascross-correction (Neufeld et al. (1970) Science 169:141-146).

A group of metabolic disorders known as lysosomal storage diseases (LSD)includes over forty genetic disorders, many of which involve geneticdefects in various lysosomal hydrolases. Representative lysosomalstorage diseases and the associated defective enzymes are listed inTable 1.

TABLE 1 Lysosomal storage disease Defective enzymeAspartylglucosaminuria Aspartylglucosaminidase Fabryalpha.-Galactosidase A Infantile Batten Disease* Palmitoyl ProteinThioesterase (CNL1) Classic Late Infantile Batten Tripeptidyl PeptidaseDisease* (CNL2) Juvenile Batten Disease* Lysosomal Transmembrane Protein(CNL3) Batten, other forms* (CNL4- Multiple gene products CNL8)Cystinosis Cysteine transporter Farber Acid ceramidase Fucosidosis Acid.alpha.-L-fucosidase Galactosidosialidosis Protective protein/cathepsinA Gaucher types 1, 2*, and 3* Acid .beta.-glucosidase G.sub.M1gangliosidosis* Acid .beta.-galactosidase Hunter* Iduronate-2-sulfataseHurler-Scheie* alpha.-L-Iduronidase Krabbe* Galactocerebrosidase.alpha.-Mannosidosis* Acid .alpha.-mannosidase. beta.-Mannosidosis* Acid.beta.-mannosidase Maroteaux-Lamy Arylsulfatase B Metachromaticleukodystrophy* Arylsulfatase A Morquio AN-Acetylgalactosamine-6-sulfate Morquio B Acid .beta.-galactosidaseMucolipidosis II/III* N-Acetylglucosamine-1-phospho- transferaseNiemann-Pick A*, B Acid sphingomyelinase (aSM) Niemann-Pick C* NPC-1Pompe* Acid .alpha.-glucosidase Sandhoff* .beta.-Hexosaminidase BSanfilippo A* Heparan N-sulfatase Sanfilippo B*.alpha.-N-Acetylglucosaminidase Sanfilippo C* Acetyl-CoA:alpha.-glucosaminide Sanfilippo D* N-Acetylglucosamine-6-sulfateSchindler Disease* .alpha.-N-Acetylgalactosaminidase Schindler-Kanzaki.alpha.-N-Acetylgalactosaminidase Sialidosis .alpha.-Neuramidase Sly*.beta.-Glucuronidase Tay-Sachs* .beta.-Hexosaminidase A Wolman* AcidLipase *CNS involvement

The hallmark feature of LSD is the abnormal accumulation of metabolitesin the lysosomes which leads to the formation of large numbers ofdistended lysosomes in the perikaryon. A major challenge to treating LSD(as opposed to treating a liver-specific enzymopathy) is the need toreverse lysosomal storage pathology in multiple separate tissues. SomeLSDs can be effectively treated by intravenous infusion of the missingenzyme, known as enzyme replacement therapy (ERT). For example, Gauchertype 1 patients have only visceral disease and respond favorably to ERTwith recombinant glucocerebrosidase (Cerezyme™, Genzyme Corp.). However,patients with metabolic disease that affects the CNS (e.g., type 2 or 3Gaucher disease) respond partially to intravenous ERT because thereplacement enzyme is prevented from entering the brain by the bloodbrain barrier (BBB). Furthermore, attempts to introduce a replacementenzyme into the brain by direct injection have been limited in part dueto enzyme cytotoxicity at high local concentrations and limitedparenchymal diffusion rates in the brain (Partridge, Peptide DrugDelivery to the Brain, Raven Press, 1991).

One exemplary LSD, is Niemann-Pick disease type A (NPA). According toUniProtKB/Swiss-Prot entry P17405, defects in the SMPD1 gene, located onchromosome 11, (11p15.4-p15.1), are the cause of Niemann-Pick diseasetype A (NPA), also referred to as the classical infantile form.Niemann-Pick disease is a clinically and genetically heterogeneousrecessive disorder. It is caused by the accumulation of sphingomyelinand other metabolically related lipids in the lysosomes, resulting inneurodegeneration starting from early life. Patients may show xanthomas,pigmentation, hepatosplenomegaly, lymphadenopathy and mentalretardation. Niemann-Pick disease occurs more frequently amongindividuals of Ashkenazi Jewish ancestry than in the general population.NPA is characterized by very early onset in infancy and a rapidlyprogressive course leading to death by three years. The acidsphingomyelinase enzyme (aSM) converts sphingomyelin to ceramide. aSMalso has phospholipase C activities toward1,2-diacylglycerolphosphocholine and 1,2-diacylglycerolphosphoglycerol.The enzyme converts

-   -   Sphingomyelin+H₂O→N-acylsphingosine+choline phosphate.

Lysosomal storage diseases can be treated using intraventriculardelivery of the enzyme which is etiologically deficient in the disease.The administration can be performed slowly to achieve maximum effect.Effects are seen on both sides of the blood-brain barrier, making this auseful delivery means for lysosomal storage diseases which affect thebrain and/or visceral organs.

However, a need still exists for additional compositions and methods totreat lysosomal storage diseases (LSDs) in primate subjects includinghuman patients. This invention satisfies this need and provides relatedadvantages as well.

This invention provides methods and compositions to deliver a transgeneto the CNS and/or affected visceral organs of a subject byintraventricular administration of a recombinant neurotrophic viralvector containing a transgene encoding an enzyme which is defective in alysosomal storage disease. The viral delivery may be under conditionsthat favor expression of the transgene in ependymal cells.

This invention provides methods and compositions to deliver a transgeneto the CNS and/or affected visceral organs of a subject byintraventricular administration of a recombinant neurotrophic viralvector comprising a transgene encoding one or more enzymes selected fromthe group consisting of Aspartylglucosaminidase, alpha.-Galactosidase A,Palmitoyl Protein Thioesterase, Tripeptidyl Peptidase, LysosomalTransmembrane Protein, Multiple gene products, Cysteine transporter,Acid ceramidase, Acid .alpha.-L-fucosidase, Protective protein/cathepsinA, Acid .beta.-glucosidase, Acid .beta.-galactosidase,Iduronate-2-sulfatase, alpha.-L-Iduronidase, Galactocerebrosidase Acid.alpha.-mannosidase, Acid .beta.-mannosidase, Arylsulfatase B,Arylsulfatase A, N-Acetylgalactosamine-6-sulfate, Acid.beta.-galactosidase, N-Acetylglucosamine-1-, Acid sphingomyelinase,NPC-1, .alpha.-glucosidase, .beta.-Hexosaminidase B, HeparanN-sulfatase, .alpha.-N-Acetylglucosaminidase, Acetyl-CoA:alpha.-glucosaminide, N-Acetylglucosamine-6-sulfate,.alpha.-N-Acetylgalactosaminidase, alpha.-N-Acetylgalactosaminidase,.alpha.-Neuramidase, .beta.-Glucuronidase, .beta.-Hexosaminidase A, andAcid Lipase. The viral delivery may be under conditions that favorexpression of the transgene in ependymal cells.

In a further aspect, the invention provides compositions and method toameliorate the symptoms of a lysosomal storage disease in a subject byadministering a recombinant neurotrophic viral vector containing thetherapeutic transgene to the subject's brain and under conditions thatfavor expression of the transgene in a therapeutically effective amount.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows green fluorescent protein (GFP) expression in mice treatedwith AAV4-GFP. GFP is distributed in the ependymal cell layer of theventricular system following intraventricular delivery of AAV4-GFP.

FIG. 2 shows green fluorescent protein (GFP) expression in mice treatedwith AAV4-GFP. GFP is distributed in the ependymal cell layer of thespinal cord central canal following intraventricular delivery ofAAV4-GFP.

FIG. 3 is a Kaplan-Meier survival curve demonstrating the survival dataof the AAV-GC treated K2 mice (AAV2/5 GC; represented by triangles) andthe AAV-GC treated wild-type littermate control group (WT; representedby upside-down triangles). Historical survival data from the K2 mice inthe mouse colony has been added to the Kaplan-Meier survival curve(Untreated; represented by boxes). The difference between the historicalmedian survival time of untreated K2 mice and the AAV-GC treated K2 micemedian survival time is statistically significant (p<0.0001).

FIG. 4 presents GC activity in the brain and liver of AAV-GC treated K2mice (injected as described above) as compared to several controlgroups. The first control group contained the “wild-type” mouse group,which have no disruptions in the GC gene and thus normal, endogenous GCexpression. This group was treated with AAV-GC in the same manner as thetreated K2 mice (labeled AAV5 WT in FIG. 4). The second control groupcontained the “heterozygote” mouse group, which have one disrupted copyof the GC gene and one non-disrupted copy of the GC gene and thusexpress some endogenous level of GC. These mice were also treated withAAV-GC (labeled AAV5 Het in FIG. 4). The final control group alsocontained the “wild-type” mouse group, but these mice were not treatedwith AAV-GC (labeled WT in FIG. 4).

FIG. 5 represents the growth of AAV-GC treated K2 mice over time (asmeasured by weight) as compared to the growth over time of AAV-GCtreated wild-type littermates. In this mouse model and regardless ofgenotype, the mouse pups from smaller litters tend to be larger than themouse pups from larger litters. To account for this variation in mousepup size as a function of the litter size, the weight of the K2 mousepups are normalized as a percentage of their own littermates.

DETAILED DESCRIPTION OF THE INVENTION

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of immunology, molecular biology,microbiology, cell biology and recombinant DNA, which are within theskill of the art. See, e.g., Sambrook, Fritsch and Maniatis, MOLECULARCLONING: A LABORATORY MANUAL, 2^(nd) edition (1989); CURRENT PROTOCOLSIN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); the seriesMETHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICALAPPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMALCELL CULTURE (R. I. Freshney, ed. (1987)).

As used in the specification and claims, the singular form “a,” “an,”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants from the isolation and purification method andpharmaceutically acceptable carriers, such as phosphate buffered saline,preservatives, and the like. “Consisting of” shall mean excluding morethan trace elements of other ingredients and substantial method stepsfor administering the compositions of this invention. Embodimentsdefined by each of these transition terms are within the scope of thisinvention.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 0.1. It is to be understood, althoughnot always explicitly stated that all numerical designations arepreceded by the term “about.” It also is to be understood, although notalways explicitly stated, that the reagents described herein are merelyexemplary and that equivalents of such are known in the art.

The term “transgene” refers to a polynucleotide that is introduced intoa cell of and is capable of being transcribed into RNA and optionally,translated and/or expressed under appropriate conditions. In one aspect,it confers a desired property to a cell into which it was introduced, orotherwise leads to a desired therapeutic or diagnostic outcome.

The terms “genome particles (gp),” or “genome equivalents,” or “genomecopies” (gc), as used in reference to a viral titer, refer to the numberof virions containing the recombinant AAV DNA genome, regardless ofinfectivity or functionality. The number of genome particles in aparticular vector preparation can be measured by procedures such asdescribed in the Examples herein, or for example, in Clark et al. (1999)Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther.,6:272-278.

The terms “infection unit (iu),” “infectious particle,” or “replicationunit,” as used in reference to a viral titer, refer to the number ofinfectious and replication-competent recombinant AAV vector particles asmeasured by the infectious center assay, also known as replicationcenter assay, as described, for example, in McLaughlin et al. (1988) J.Virol., 62:1963-1973.

The term “transducing unit (tu)” as used in reference to a viral titer,refers to the number of infectious recombinant AAV vector particles thatresult in the production of a functional transgene product as measuredin functional assays such as described in Examples herein, or forexample, in Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or inFisher et al. (1996) J. Virol., 70:520-532 (LFU assay).

The terms “therapeutic,” “therapeutically effective amount,” and theircognates refer to that amount of an RNA, DNA or expression product ofDNA and/or RNA that results in prevention or delay of onset oramelioration of symptoms of in a subject or an attainment of a desiredbiological outcome, such as correction of storage pathology, e.g.,cellular pathology associated with substrate accumulation in a patientwith a lysosomal storage disease. The term “therapeutic correction”refers to that degree of correction which results in prevention or delayof onset or amelioration of symptoms in a subject. The effective amountcan be determined by known empirical methods.

A “composition” is also intended to encompass a combination of activeagent and another carrier, e.g., compound or composition, inert (forexample, a detectable agent or label) or active, such as an adjuvant,diluent, binder, stabilizer, buffers, salts, lipophilic solvents,preservative, adjuvant or the like. Carriers also include pharmaceuticalexcipients and additives proteins, peptides, amino acids, lipids, andcarbohydrates (e.g., sugars, including monosaccharides, di-, tri-,tetra-, and oligosaccharides; derivatized sugars such as alditols,aldonic acids, esterified sugars and the like; and polysaccharides orsugar polymers), which can be present singly or in combination,comprising alone or in combination 1-99.99% by weight or volume.Exemplary protein excipients include serum albumin such as human serumalbumin (HSA), recombinant human albumin (rHA), gelatin, casein, and thelike. Representative amino acid/antibody components, which can alsofunction in a buffering capacity, include alanine, glycine, arginine,betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine,leucine, isoleucine, valine, methionine, phenylalanine, aspartame, andthe like. Carbohydrate excipients are also intended within the scope ofthis invention, examples of which include but are not limited tomonosaccharides such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitolsorbitol (glucitol) and myoinositol.

The term carrier further includes a buffer or a pH adjusting agent;typically, the buffer is a salt prepared from an organic acid or base.Representative buffers include organic acid salts such as salts ofcitric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid,succinic acid, acetic acid, or phthalic acid; Tris, tromethaminehydrochloride, or phosphate buffers. Additional carriers includepolymeric excipients/additives such as polyvinylpyrrolidones, ficolls (apolymeric sugar), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-.quadrature.-cyclodextrin), polyethylene glycols,flavoring agents, antimicrobial agents, sweeteners, antioxidants,antistatic agents, surfactants (e.g., polysorbates such as “TWEEN 20”and “TWEEN 80”), lipids (e.g., phospholipids, fatty acids), steroids(e.g., cholesterol), and chelating agents (e.g., EDTA).

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agents.The compositions also can include stabilizers and preservatives and anyof the above noted carriers with the additional provision that they beacceptable for use in vivo. For examples of carriers, stabilizers andadjuvants, see Martin REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co.,Easton (1975) and Williams & Williams, (1995), and in the “PHYSICIAN'SDESK REFERENCE”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998).Carriers may also comprise artificial cerebrospinal fluid (aCSF).

A “subject,” “individual” or “patient” is used interchangeably herein,which refers to a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, murines, rats, simians,humans, farm animals, sport animals, and pets.

A “control” is an alternative subject or sample used in an experimentfor comparison purpose. A control can be “positive” or “negative.” Forexample, where the purpose of the experiment is to determine acorrelation of an altered expression level of a gene with a particulartype of pathology, it is generally preferable to use a positive control(a subject or a sample from a subject, carrying such alteration andexhibiting symptoms characteristic of that disease), and a negativecontrol (a subject or a sample from a subject lacking the alteredexpression and clinical symptom of that disease).

As used herein, the term “modulate” means to vary the amount orintensity of an effect or outcome, e.g., to enhance, augment, diminishor reduce.

As used herein the term “ameliorate” is synonymous with “alleviate” andmeans to reduce or lighten. For example one may ameliorate the symptomsof a disease or disorder by making them more bearable.

For identification of structures in the human brain, see, e.g., TheHuman Brain: Surface, Three-Dimensional Sectional Anatomy With MRI, andBlood Supply, 2nd ed., eds. Deuteron et al., Springer Vela, 1999; Atlasof the Human Brain, eds. Mai et al., Academic Press; 1997; and Co-PlanarStereotaxic Atlas of the Human Brain: 3-Dimensional Proportional System:An Approach to Cerebral Imaging, eds. Tamarack et al., Thyme MedicalPub., 1988. For identification of structures in the mouse brain, see,e.g., The Mouse Brain in Stereotaxic Coordinates, 2nd ed., AcademicPress, 2000.

In aspects where gene transfer is mediated by a DNA viral vector, suchas an adenovirus (Ad) or adeno-associated virus (AAV), a vectorconstruct refers to the polynucleotide comprising the viral genome orpart thereof, and a transgene. Adenoviruses (Ads) are a relatively wellcharacterized, homogenous group of viruses, including over 50 serotypes.See, e.g., International PCT Application No. WO 95/27071. Ads are easyto grow and do not require integration into the host cell genome.Recombinant Ad derived vectors, particularly those that reduce thepotential for recombination and generation of wild-type virus, have alsobeen constructed. See, International PCT Application Nos. WO 95/00655and WO 95/11984. Wild-type AAV has high infectivity and specificityintegrating into the host cell's genome. See, Hermonat and Muzyczka(1984) Proc. Natl. Acad. Sci. USA 81:6466-6470 and Lebkowski, et al.(1988) Mol. Cell. Biol. 8:3988-3996.

In one aspect, the invention provides a method to deliver a transgene tothe brain of a subject by intraventricular administration of arecombinant neurotrophic viral vector containing a transgene encoding anenzyme which is defective in a LSD. The delivery is under conditionsthat favor expression of the transgene in ependymal cells.

In another aspect, the invention provides a method of delivering atherapeutic transgene product to the CNS in a mammal afflicted with alysosomal storage disease, where the transgene may encode a lysosomalenzyme. The transgene can be administered via a neurotrophic virus. Thevirus can be administered via the ventricles. Ependymal cells may betransduced to express the transgene and secrete the encoded proteinproduct.

In an alternate embodiment, the invention is a method to treat alysosomal storage disease in a subject by intraventricularadministration of a recombinant neurotrophic viral vector containing atherapeutic transgene to the brain of the subject, wherein the transgeneis expressed in a therapeutically effective amount in the subject.

This invention also is a method to ameliorate the symptoms of alysosomal storage disease in a subject by intraventricularadministration of a recombinant neurotrophic viral vector containing atherapeutic transgene to the brain, wherein said transgene is expressedin a therapeutically effective amount in the subject.

Suitable neurotrophic viral vectors for the practice of this inventioninclude, but are not limited to adeno-associated viral vectors (AAV),herpes simplex viral vectors (U.S. Pat. No. 5,672,344) and lentiviralvectors.

In the methods of the invention, AAV of any serotype can be used. Theserotype of the viral vector used in certain embodiments of theinvention is selected from the group consisting from AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, and AAV8 (see, e.g., Gao et al. (2002) PNAS,99:11854-11859; and Viral Vectors for Gene Therapy: Methods andProtocols, ed. Machida, Humana Press, 2003). Other serotype besidesthose listed herein can be used. Furthermore, pseudotyped AAV vectorsmay also be utilized in the methods described herein. Pseudotyped AAVvectors are those which contain the genome of one AAV serotype in thecapsid of a second AAV serotype; for example, an AAV vector thatcontains the AAV2 capsid and the AAV1 genome or an AAV vector thatcontains the AAV5 capsid and the AAV 2 genome (Auricchio et al., (2001)Hum. Mol. Genet., 10(26):3075-81).

AAV vectors are derived from single-stranded (ss) DNA parvoviruses thatare nonpathogenic for mammals (reviewed in Muzyscka (1992) Curr. Top.Microb. Immunol., 158:97-129). Briefly, recombinant AAV-based vectorshave the rep and cap viral genes that account for 96% of the viralgenome removed, leaving the two flanking 145-basepair (bp) invertedterminal repeats (ITRs), which are used to initiate viral DNAreplication, packaging and integration. In the absence of helper virus,wild-type AAV integrates into the human host-cell genome withpreferential site-specificity at chromosome 19q 13.3 or it may bemaintained episomally. A single AAV particle can accommodate up to 5 kbof ssDNA, therefore leaving about 4.5 kb for a transgene and regulatoryelements, which is typically sufficient. However, trans-splicing systemsas described, for example, in U.S. Pat. No. 6,544,785, may nearly doublethis limit.

In an illustrative embodiment, AAV is AAV4. Adeno-associated virus ofmany serotypes, especially AAV2, have been extensively studied andcharacterized as gene therapy vectors. Those skilled in the art will befamiliar with the preparation of functional AAV-based gene therapyvectors. Numerous references to various methods of AAV production,purification and preparation for administration to human subjects can befound in the extensive body of published literature (see, e.g., ViralVectors for Gene Therapy: Methods and Protocols, ed. Machida, HumanaPress, 2003). Additionally, AAV-based gene therapy targeted to cells ofthe CNS has been described in U.S. Pat. Nos. 6,180,613 and 6,503,888.Additional exemplary AAV vectors are recombinant AAV2/1, AAV2/2, AAV2/5,AAV2/7 and AAV2/8 serotype vectors encoding human protein.

In certain methods of the invention, the vector comprises a transgeneoperably linked to a promoter. The transgene encodes a biologicallyactive molecule, expression of which in the CNS results in at leastpartial correction of storage pathology and/or stabilization of diseaseprogression. The transgene may encode one or more ofAspartylglucosaminidase, alpha.-Galactosidase A, Palmitoyl ProteinThioesterase, Tripeptidyl Peptidase, Lysosomal Transmembrane Protein,Multiple gene products, Cysteine transporter, Acid ceramidase, Acid.alpha.-L-fucosidase, Protective protein/cathepsin A, Acid.beta.-glucosidase, Acid .beta.-galactosidase, Iduronate-2-sulfatase,alpha.-L-Iduronidase, Galactocerebrosidase, Acid .alpha.-mannosidase,Acid .beta.-mannosidase, Arylsulfatase B, Arylsulfatase A,N-Acetylgalactosamine-6-sulfate, Acid .beta.-galactosidase,N-Acetylglucosamine-1-, Acid sphingomyelinase, NPC-1,.alpha.-glucosidase, .beta.-Hexosaminidase B, Heparan N-sulfatase,.alpha.-N-Acetylglucosaminidase, Acetyl-CoA: alpha.-glucosaminide,N-Acetylglucosamine-6-sulfate, .alpha.-N-Acetylgalactosaminidase,alpha.-N-Acetylgalactosaminidase, .alpha.-Neuramidase,.beta.-Glucuronidase, .beta.-Hexosaminidase A, and Acid Lipase.

One particularly useful enzyme for treating Niemann-Pick A or B is acidsphingomyelinase (aSM), such as that shown in SEQ ID NO: 1. (Residues1-46 constitute the signal sequence which is cleaved upon secretion.)One particularly useful enzyme for treating Gaucher disease isglucocerebrosidase. One particularly useful enzyme for treating MPS Idisease is alpha-L-Iduronidase. One particularly useful enzyme fortreating MPS II disease is iduronate-2-sulfatase. One particularlyuseful enzyme for treating Pompe disease, or glycogen storage diseasetype II (GSDII), also termed acid maltase deficiency (AMD) is acidalpha-glucosidase. One particularly useful enzyme for treating classiclate infantile Batten disease (CLN2) is tripeptidyl peptidase. Theseenzymes are often recombinant forms of the enzymes produced usingmethods well-known in the art. In one embodiment, the enzyme is arecombinant human enzyme.

Although a particular amino acid sequence is shown in SEQ ID NO: 1,normal variants in the human population which retain activity can beused as well. Typically these normal variants differ by just one or tworesidues from the sequence shown in SEQ ID NO: 1. The variants to beused should be at least 95%, 96%, 97%, 98%, or 99% identical to SEQ IDNO: 1. Variants which are associated with disease or reduced activityshould not be used. Typically the mature form of the enzyme will bedelivered. This will begin with residue 47 as shown in SEQ ID NO: 1. Ina similar manner, normal variants in the human population of such LSDenzymes as glucocerebrosidase, alpha-L-Iduronidase,iduronate-2-sulfatase, acid alpha-glucosidase, and tripeptidyl peptidasethat which retain enzymatic activity can be used as well.

The populations treated by the methods of the invention include, but arenot limited to, patients having or at risk for developing aneurometabolic disorder, e.g., an LSD, such as diseases listed in Table1, particularly, if such a disease affects the CNS and visceral organs.In an illustrative embodiment, the disease is Niemann-Pick disease, typeA, B, or C, Gaucher disease, Mucopolysaccharidoses (MPS), Pompe disease,or Batten disease.

A transgene encoding aSM or other lysosomal hydrolase enzyme can beincorporated into a pharmaceutical composition useful to treat, e.g.,inhibit, attenuate, prevent, or ameliorate, a condition characterized byan insufficient level of a lysosomal hydrolase activity. Thepharmaceutical composition will be administered to a subject sufferingfrom a lysosomal hydrolase deficiency or someone who is at risk ofdeveloping said deficiency. The compositions should contain atherapeutic or prophylactic amount of the transgene encoding an aSM orother lysosomal hydrolase enzyme, in a pharmaceutically-acceptablecarrier. The pharmaceutical carrier can be any compatible, non-toxicsubstance suitable to deliver the polypeptides to the patient. Sterilewater, alcohol, fats, and waxes may be used as the carrier.Pharmaceutically-acceptable adjuvants, buffering agents, dispersingagents, and the like, may also be incorporated into the pharmaceuticalcompositions. The carrier can be combined with the aSM or otherlysosomal hydrolase enzyme in any form suitable for administration byintraventricular injection or infusion (also possibly intravenous orintrathecal) or otherwise. Suitable carriers include, for example,physiological saline, bacteriostatic water, Cremophor EL™ (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS), other salinesolutions, dextrose solutions, glycerol solutions, water and oilsemulsions such as those made with oils of petroleum, animal, vegetable,or synthetic origin (peanut oil, soybean oil, mineral oil, or sesameoil). An artificial CSF can be used as a carrier. The carrier willpreferably be sterile and free of pyrogens. The concentration of thetransgene encoding aSM or other lysosomal hydrolase enzyme in thepharmaceutical composition can vary widely.

Intracerebroventricular, or intraventricular delivery of a recombinantviral vector may be performed in any one or more of the brain'sventricles, which are filled with cerebrospinal fluid (CSF). CSF is aclear fluid that fills the ventricles, is present in the subarachnoidspace, and surrounds the brain and spinal cord. CSF is produced by thechoroid plexuses and via the weeping or transmission of tissue fluid bythe brain into the ventricles. The choroid plexus is a structure liningthe floor of the lateral ventricle and the roof of the third and fourthventricles. Certain studies have indicated that these structures arecapable of producing 400-600 ccs of fluid per day consistent with anamount to fill the central nervous system spaces four times in a day. Inadults, the volume of this fluid has been calculated to be from 125 to150 ml (4-5 oz). The CSF is in continuous formation, circulation andabsorption. Certain studies have indicated that approximately 430 to 450ml (nearly 2 cups) of CSF may be produced every day. Certaincalculations estimate that production equals approximately 0.35 ml perminute in adults and 0.15 per minute in infants. The choroid plexuses ofthe lateral ventricles produce the majority of CSF. It flows through theforamina of Monro into the third ventricle where it is added to byproduction from the third ventricle and continues down through theaqueduct of Sylvius to the fourth ventricle. The fourth ventricle addsmore CSF; the fluid then travels into the subarachnoid space through theforamina of Magendie and Luschka. It then circulates throughout the baseof the brain, down around the spinal cord and upward over the cerebralhemispheres.

While not wishing to be bound by any theory or mechanism of operation,administration to the CNS of an afflicted subject of a neurotrophicviral vector carrying a transgene encoding a therapeutic product permitstransport of the viral vector and/or expressed product via the CSFthroughout the CNS and/or to the visceral organs. The CSF empties intothe blood via the arachnoid villi and intracranial vascular sinuses,thereby delivering the enzymes and/or the transgenes to the visceralorgans that are known to be affected in LSDs. The visceral organs whichare often affected in Niemann-Pick disease, for example, are the lungs,spleen, kidney, and liver. The gene therapy provides diminished amountsof substrate in at least the brain and potentially in visceral organs.The reduction in substrate accumulated in the brain, lungs, spleen,kidney, and/or liver may be dramatic. Reductions of greater that 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% can be achieved. The reductionachieved is not necessarily uniform from patient to patient or even fromorgan to organ within a single patient.

The lysosomal storage diseases (LSD) include over forty geneticdisorders, many of which involve genetic defects in various lysosomalhydrolases. Representative lysosomal storage diseases and the associateddefective enzymes are listed in Table 1.

Gaucher disease results as a consequence of an inherited deficiency ofthe lysosomal hydrolase glucocerebrosidase (GC), leading to theaccumulation of its substrate, glucosylceramide (GL-1), in the lysosomesof histiocytes. The progressive accumulation of GL-1 in tissuemacrophages (Gaucher cells) occurs in various tissues. The extent of theaccumulation is dependent in part on the genotype. Clinically, threedifferent Gaucher phenotypes are recognized, the non-neuropathic type 1,which is the most common with onset ranging from early childhood toadulthood, and the neuropathic types 2 and 3, presenting in infancy andearly childhood, respectively. The primary clinical manifestationscommon to all forms of Gaucher disease are hepatosplenomegaly,cytopenia, pathological bone fractures and, occasionally, pulmonaryfailure. A detailed discussion of Gaucher disease may be found in theOnline Metabolic & Molecular Bases of Inherited Diseases, Part 16,Chapter 146 and 146.1 (2007). In patients with type 2 and type 3 Gaucherdisease in whom there is significant central nervous system involvement,intraventricular delivery of the defective LSD enzyme leads to improvedmetabolic status of the brain and possibly the affected visceral(non-CNS) organs. Intraventricular delivery of the defective LSD enzymein subjects with Gaucher type 1 disease leads to improved metabolicstatus of affected visceral (non-CNS) organs. There are animal models ofGaucher disease, which have derived from mouse models created bytargeted disruption of the corresponding mouse gene. For example, aGaucher mouse model harboring the D409V mutation in the mouse GC locusexists (Xu, Y-H et al. (2003). Am. J. Pathol. 163:2093-2101). Theheterozygous mouse, gbaD409V/null, exhibits □5% of normal GC activity invisceral tissues and develops lipid-engorged macrophages (Gaucher cells)in the liver, spleen, lung and bone marrow by 4 months of age. Otherexamples of mouse models of Gaucher disease are the models of acuteneuronopathic Gaucher disease developed by Enquist et al. 2007 PNAS104(44): 17483-17488. These mice exhibit close similarity, in bothpathological findings and clinical manifestations, to human patientswith severe neuronopathic Gaucher disease. Accordingly, all of themodels discussed above are suitable systems in which to evaluate thebenefits of intraventricular delivery of a viral neurotrophic vectorencoding the defective LSD enzyme in subjects with Gaucher disease.

Niemann-Pick disease (NPD) is a lysosomal storage disease and is aninherited neurometabolic disorder characterized by a genetic deficiencyin acid sphingomyelinase (aSM; sphingomyelin cholinephosphohydrolase, EC3.1.3.12). The lack of functional aSM protein results in theaccumulation of sphingomyelin substrate within the lysosomes of neuronsand glia throughout the brain. This leads to the formation of largenumbers of distended lysosomes in the perikaryon, which are a hallmarkfeature and the primary cellular phenotype of type A NPD. The presenceof distended lysosomes correlates with the loss of normal cellularfunction and a progressive neurodegenerative course that leads to deathof the affected individual in early childhood (The Metabolic andMolecular Bases of Inherited Diseases, eds. Scriver et al., McGraw-Hill,New York, 2001, pp. 3589-3610). Secondary cellular phenotypes (e.g.,additional metabolic abnormalities) are also associated with thisdisease, notably the high level accumulation of cholesterol in thelysosomal compartment. Sphingomyelin has strong affinity forcholesterol, which results in the sequestering of large amounts ofcholesterol in the lysosomes of aSMKO mice and human patients (Leventhalet al. (2001) J. Biol. Chem., 276:44976-44983; Slotte (1997) Subcell.Biochem., 28:277-293; and Viana et al. (1990) J. Med. Genet.,27:499-504.) A detailed discussion of NPD disease may be found in theOnline Metabolic & Molecular Bases of Inherited Diseases, Part 16,Chapter 144 (2007). There are animal models of NPD. For example, aSMKOmice are an accepted model of types A and B Niemann-Pick disease(Horinouchi et al. (1995) Nat. Genetics, 10:288-293; Jin et al. (2002)J. Clin. Invest., 109:1183-1191; and Otterbach (1995) Cell,81:1053-1061). Intraventricular delivery of a transgene encoding thedefective LSD enzyme leads to improved metabolic status of the brain andthe affected visceral (non-CNS) organs.

Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorderscaused by deficiencies of enzymes catalyzing the degradation ofglycosaminoglycans (mucopolysaccharides). There are 11 known enzymedeficiencies that give rise to 7 distinct MPS, including MPS I (Hurler,Scheie, and Hurler-Scheie Syndromes) and MPS II (Hunter Syndrome). Adetailed discussion of MPS may be found in the Online Metabolic &Molecular Bases of Inherited Diseases, Part 16, Chapter 136 (2007).There are numerous animal models of MPS, which have derived fromnaturally occurring mutations in dogs, cats, rats, mice, and goats, aswell as mouse models created by targeted disruption of the correspondingmouse gene. The biochemical and metabolic features of these animalmodels are generally quite similar to those found in humans; however,the clinical presentations may be milder. For example, accepted modelsfor MPS I include a murine model [Clark, L A et al., Hum. Mol. Genet.(1997), 6:503] and a canine model [Menon, K P et al., Genomics (1992),14:763. For example, accepted models for MPS II include a mouse model[Muenzer, J. et al., (2002), Acta Paediatr. Suppl.;91(439):98-9]. In theMPS that have central nervous system involvement, such as is found inpatients with MPS I and MPS II, intraventricular delivery of a transgeneencoding the defective LSD enzyme leads to improved metabolic status ofthe brain and possibly the affected visceral (non-CNS) organs.

Pompe disease, or glycogen storage disease type II (GSDII), also termedacid maltase deficiency (AMD) is an inherited disorder of glycogenmetabolism resulting from defects in activity of the lysosomal hydrolaseacid alpha-glucosidase in all tissues of affected individuals. Theenzyme deficiency results in intralysosomal accumulation of glycogen ofnormal structure in numerous tissues. The accumulation is most marked incardiac and skeletal muscle and in hepatic tissues of infants with thegeneralized disorder. In late-onset GSDII, intralysosomal accumulationof glycogen is virtually limited to skeletal muscle and is of lessermagnitude. Electromyographic abnormalities suggestive of the diagnosisinclude pseudomyotonic discharges and irritability, but in juvenile- andadult-onset patients, the abnormalities can vary in different muscles.CAT scans can reveal the site(s) of affected muscles. Most patients haveelevated blood plasma levels of creatine kinase (CK) and elevations inhepatic enzymes, particularly in adult-onset patients, can be found.There are several naturally occurring animal models of the infantile-and late-onset disease. There is a knockout mouse model [Bijvoet A G etal., Hum. Mol. Genet. (1998); 7:53-62.]. Ameliorative effects of enzymetherapy have been described in knockout mice [Raben, N et al., Mol.Genet. Metab. (2003); 80:159-69] and in a quail model. Intraventriculardelivery of a transgene encoding the defective LSD enzyme leads toimproved metabolic status of the brain and possibly the affectedvisceral (non-CNS) organs.

The neuronal ceroid lipofuscinoses (NCL) are a group ofneurodegenerative disorders distinguished from other neurodegenerativediseases by the accumulation of autofluorescent material (“agingpigment”) in the brain and other tissues. The major clinical featuresinclude seizures, psychomotor deterioration, blindness, and prematuredeath. Distinct subgroups of NCL have been recognized that differ in theage of onset of symptoms and the appearance of the storage material byelectron microscopy. Three major groups-infantile (INCL), classical lateinfantile (LINCL), and juvenile (JNCL, also referred to as Battendisease)—are caused by autosomal recessive mutations in the CLN1, CLN2,and CLN3 genes, respectively. The protein products of the CLN1(palmitoyl-protein thioesterase) and CLN2 (tripeptidyl peptidase orpepinase) genes are soluble lysosomal enzymes, whereas the CLN3 protein(battenin) is a lysosomal membrane protein, as is (tentatively) the CLN5protein. The identification of mutations in genes encoding lysosomalproteins in several forms of NCL has led to the recognition of thelipofuscinoses as true lysosomal storage disorders. A detaileddiscussion of NCL disease may be found in the Online Metabolic &Molecular Bases of Inherited Diseases, Part 16, Chapter 154 (2007).Naturally occurring NCL disorders have been described in the sheep, dog,and mouse models have been derived by targeted disruption of acorresponding mouse gene [see e.g., Katz, M L et al., J. Neurosci. Res.(1999); 57:551-6; Cho, S K et al., Glycobiology (2005); 15:637-48.]Intraventricular delivery of a transgene encoding the defective LSDenzyme leads to improved metabolic status of the brain and possibly theaffected visceral (non-CNS) organs.

A detailed discussion of additional lysosomal storage disordersdisclosed in Table 1, in which intraventricular delivery of thedefective LSD enzyme in the disease, may be found in the OnlineMetabolic & Molecular Bases of Inherited Diseases, Part 16 (2007).

The level of transgene expression in eukaryotic cells is largelydetermined by the transcriptional promoter within the transgeneexpression cassette. Promoters that show long-term activity and aretissue- and even cell-specific are used in some embodiments. Nonlimiting examples of promoters include, but are not limited to, thecytomegalovirus (CMV) promoter (Kaplitt et al. (1994) Nat. Genet.8:148-154), CMV/human β3-globin promoter (Mandel et al. (1998) J.Neurosci. 18:4271-4284), GFAP promoter (Xu et al. (2001) Gene Ther.8:1323-1332), the 1.8-kb neuron-specific enolase (NSE) promoter (Kleinet al. (1998) Exp. Neurol. 150:183-194), chicken beta actin (CBA)promoter (Miyazaki (1989) Gene 79:269-277), the β-glucuronidase (GUSB)promoter (Shipley et al. (1991) Genetics 10:1009-1018), and ubiquitinpromoters such as those isolated from human ubiquitin A, human ubiquitinB, and human ubiquitin C as described in U.S. Pat. No. 6,667,174. Toprolong expression, other regulatory elements may additionally beoperably linked to the transgene, such as, e.g., the Woodchuck HepatitisVirus Post-Regulatory Element (WPRE) (Donello et al. (1998) J. Virol.72:5085-5092) or the bovine growth hormone (BGH) polyadenylation site.

For some CNS gene therapy applications, it may be necessary to controltranscriptional activity. To this end, pharmacological regulation ofgene expression with viral vectors can been obtained by includingvarious regulatory elements and drug-responsive promoters as described,for example, in Haberman et al. (1998) Gene Ther. 5:1604-16011; and Yeet al. (1995) Science 283:88-91.

In certain embodiments, the concentration or titer of the vector in thecomposition is at least: (a) 5, 6, 7, 8, 9, 10, 15, 20, 25, or 50 (×10¹²gp/ml); (b) 5, 6, 7, 8, 9, 10, 15, 20, 25, or 50 (×10⁹ to/ml); or (c) 5,6, 7, 8, 9, 10, 15, 20, 25, or 50 (×10¹⁰ iu/ml).

In one aspect, the transgene encodes a biologically active molecule,expression of which in the CNS results in at least partial correction ofstorage pathology and/or stabilization of disease progression. In someembodiments, the therapeutic transgene product is an aSM protein thatalleviates and/or prevents the symptoms of Niemann-Pick A and B. SeeRoaul et al. (2005) Nat. Med. 11(4):423-428 and Ralph et al. (2005) Nat.Med. 11(4):429-433. In other embodiments, transgenes encoding theenzymes defective in other LSD are delivered, as appropriate for theparticular patient.

In one aspect when performing these methods, the transgene expresses atherapeutic amount of an enzyme selected from the group consisting ofAspartylglucosaminidase, alpha.-Galactosidase A, Palmitoyl ProteinThioesterase, Tripeptidyl Peptidase, Lysosomal Transmembrane Protein,Multiple gene products, Cysteine transporter, Acid ceramidase, Acid.alpha.-L-fucosidase, Protective protein/cathepsin A, Acid.beta.-glucosidase, or, Acid .beta.-galactosidase,Iduronate-2-sulfatase, alpha.-L-Iduronidase, Galactocerebrosidase, Acid.alpha.-mannosidase, Acid .beta.-mannosidase, Arylsulfatase B,Arylsulfatase A, N-Acetylgalactosamine-6-sulfate, Acid.beta.-galactosidase, N-Acetylglucosamine-1-, Acid sphingomyelinase,NPC-1, .alpha.-glucosidase, .beta.-Hexosaminidase B, HeparanN-sulfatase, .alpha.-N-Acetylglucosaminidase, Acetyl-CoA:alpha.-glucosaminide, N-Acetylglucosamine-6-sulfate,.alpha.-N-Acetylgalactosaminidase, alpha.-N-Acetylgalactosaminidase,.alpha.-Neuramidase, .beta.-Glucuronidase, .beta.-Hexosaminidase A, andAcid Lipase.

For identification of structures in the human brain, see, e.g., TheHuman Brain: Surface, Three-Dimensional Sectional Anatomy With MRI, andBlood Supply, 2nd ed., eds. Deuteron et al., Springer Vela, 1999; Atlasof the Human Brain, eds. Mai et al., Academic Press; 1997; and Co-PlanarStereotaxic Atlas of the Human Brain: 3-Dimensional Proportional System:An Approach to Cerebral Imaging, eds. Tamarack et al., Thyme MedicalPub., 1988. For identification of structures in the mouse brain, see,e.g., The Mouse Brain in Stereotaxic Coordinates, 2nd ed., AcademicPress, 2000.

To deliver the solution or other composition containing the viral vectorspecifically to a particular region of the central nervous system, suchas to a particular ventricle, e.g., to the lateral ventricles or to thefourth ventricle of the brain, it may be administered by stereotaxicmicroinjection. For example, on the day of surgery, patients will havethe stereotaxic frame base fixed in place (screwed into the skull). Thebrain with stereotaxic frame base (MRI-compatible with fiduciarymarkings) will be imaged using high resolution MRI. The MRI images willthen be transferred to a computer that runs stereotaxic software. Aseries of coronal, sagittal and axial images will be used to determinethe target site of vector injection, and trajectory. The softwaredirectly translates the trajectory into 3-dimensional coordinatesappropriate for the stereotaxic frame. Burr holes are drilled above theentry site and the stereotaxic apparatus localized with the needleimplanted at the given depth. The vector solution in a pharmaceuticallyacceptable carrier will then be injected. Additional routes ofadministration may be used, e.g., superficial cortical application underdirect visualization, or other non-stereotaxic application.

One way for delivering the viral vector is to use a pump. Such pumps arecommercially available, for example, from Alzet (Cupertino, Calif.) orMedtronic (Minneapolis, Minn.). The pump may be implantable. Anotherconvenient way to administer the vector is to use a cannula or acatheter.

The subject invention provides methods to modulate, correct or reducesymptoms in a subject afflicted with a lysosomal storage disease, byreducing substrate accumulation in the CNS or affected visceral organs.For the purpose of illustration only, the subject may suffer from one ormore of Aspartylglucosaminuria, Fabry, Infantile Batten Disease (CNL1),Classic Late Infantile Batten Disease (CNL2), Juvenile Batten Disease(CNL3), Batten, other forms (CNL4-CNL8), Cystinosis, Farber,Fucosidosis, Galactosidosialidosis, Gaucher types 1, 2, and 3, G.sub.M1gangliosidosis, Hunter, Hurler-Scheie, Krabbe, alpha.-Mannosidosis,beta.-Mannosidosis, Maroteaux-Lamy, Metachromatic leukodystrophy,Morquio A, Morquio B, Mucolipidosis Niemann-Pick A, B, Niemann-Pick C,Pompe Acid, Sandhoff, Sanfilippo A, Sanfilippo B, Sanfilippo C,Sanfilippo D, Schindler Disease, Schindler-Kanzaki Sialidosis, Sly,Tay-Sachs, and Wolman Disease.

If desired, the human brain structure can be correlated to similarstructures in the brain of another mammal. For example, most mammals,including humans and rodents, show a similar topographical organizationof the entorhinal-hippocampus projections, with neurons in the lateralpart of both the lateral and medial entorhinal cortex projecting to thedorsal part or septal pole of the hippocampus, whereas the projection tothe ventral hippocampus originates primarily from neurons in medialparts of the entorhinal cortex (Principles of Neural Science, 4th ed.,eds. Kandel et al., McGraw-Hill, 1991; The Rat Nervous System, 2nd ed.,ed. Paxinos, Academic Press, 1995). Furthermore, layer II cells of theentorhinal cortex project to the dentate gyrus, and they terminate inthe outer two-thirds of the molecular layer of the dentate gyrus. Theaxons from layer III cells project bilaterally to the cornu ammonisareas CA1 and CA3 of the hippocampus, terminating in the stratumlacunose molecular layer.

In one aspect, the disclosed methods include administering to the CNS ofan afflicted subject a neurotrophic viral vector carrying a transgeneencoding a therapeutic product and allowing the transgene to beexpressed within the CNS and/or visceral organs at a level sufficient toexert a therapeutic effect as the expressed protein is transported viathe CSF throughout the CNS and/or visceral organs. In addition, thevector may comprise a polynucleotide encoding for a biologically activemolecule effective to treat the CNS disorder. Such biologically activemolecules may comprise peptides including but not limited to native ormutated versions of full-length proteins, native or mutated versions ofprotein fragments, synthetic polypeptides.

In an illustrative embodiment, the administration is accomplished bydirect injection of a high titer vector solution into one or more of theventricular spaces of the brain, such as the lateral ventricle of asubject or patient. For example, the administration is by direct bolusinjection into one or more ventricles of the brain such as the lateraland fourth ventricles.

In some embodiments, the methods comprise administration of a high titerneurotrophic vector carrying a therapeutic transgene so that thetransgene product is expressed at a therapeutic level at a first sitewithin the CNS distal to the ultimate site of action of the expressedproduct. In some embodiments, the viral titer of the composition is atleast: (a) 5, 6, 7, 8, 9, 10, 15, 20, 25, or 50 (×10¹² gp/ml); (b) 5, 6,7, 8, 9, 10, 15, 20, 25, or 50 (×10⁹ to/ml); or (c) 5, 6, 7, 8, 9, 10,15, 20, 25, or 50 (×10¹⁰ iu/ml).

In experimental mice, the total volume of injected AAV solution is forexample, between 1 to 20 μl. For other mammals, including the humanbrain, volumes and delivery rates are appropriately scaled. For example,it has been demonstrated that volumes of 150 μl can be safely injectedin the primate brain (Janson et al. (2002) Hum. Gene Ther.13:1391-1412). Treatment may consist of a single injection per targetsite, or may be repeated in one or more ventricles. Suitable ventriclesinclude the lateral ventricles, third ventricle, and the fourthventricle. Multiple injection sites can be used. For example, in someembodiments, in addition to the first administration site, a compositioncontaining a viral vector carrying a transgene is administered toanother site which can be contralateral or ipsilateral to the firstadministration site. Injections can be single or multiple, unilateral orbilateral.

High titer AAV preparations can be produced using techniques known inthe art, e.g., as described in U.S. Pat. No. 5,658,776 and Viral Vectorsfor Gene Therapy: Methods and Protocols, ed. Machida, Humana Press,2003.

The following examples provide illustrative embodiments of theinvention. One of ordinary skill in the art will recognize the numerousmodifications and variations that may be performed without altering thespirit or scope of the present invention. Such modifications andvariations are encompassed within the scope of the invention. Theexamples do not in any way limit the invention.

Examples Titration of Recombinant Vectors

AAV vector titers are measured according to genome copy number (genomeparticles per milliliter). Genome particle concentrations are based onTaqman® PCR of the vector DNA as previously reported (Clark et al.(1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther.,6:272-278).

Vectors carrying an assayable marker gene such as the β-galactosidase(Lac Z) or green fluorescent protein gene (GFP) can be titered using aninfectivity assay. Susceptible cells (e.g., HeLa, or COS cells) aretransduced with the AAV and an assay is performed to determine geneexpression such as staining of β-galactosidase vector-transduced cellswith X-gal (5-bromo-4chloro-3-indolyl-β-D-galactopyranoside) orfluorescence microscopy for GFP-transduced cells. For example, the assayis performed as follows: 4×10⁴ HeLa cells are plated in each well of a24-well culture plate using normal growth media. After attachment, i.e.,about 24 hours later, the cells are infected with Ad type 5 at amultiplicity of infection (MOI) of 10 and transduced with serialdilutions of the packaged vector and incubated at 37° C. One to threedays later, before extensive cytopathic effects are observed, theappropriate assay is performed on the cells (e.g., X-gal staining orfluorescence microscopy). If a reporter gene such as β-galactosidase isused, the cells are fixed in 2% paraformaldehyde, 0.5% glutaraldehydeand stained for β-galactosidase activity using X-gal. Vector dilutionsthat give well-separated cells are counted. Each positive cellrepresents 1 transduction unit (tu) of vector.

Animal Model

ASMKO mice are an accepted model of types A and B Niemann-Pick disease(Horinouchi et al. (1995) Nat. Genetics, 10:288-293; Jin et al. (2002)J. Clin. Invest., 109:1183-1191; and Otterbach (1995) Cell,81:1053-1061). Niemann-Pick disease (NPD) is classified as a lysosomalstorage disease and is an inherited neurometabolic disordercharacterized by a genetic deficiency in acid sphingomyelinase (ASM;sphingomyelin cholinephosphohydrolase, EC 3.1.3.12). The lack offunctional ASM protein results in the accumulation of sphingomyelinsubstrate within the lysosomes of neurons and glia throughout the brain.This leads to the formation of large numbers of distended lysosomes inthe perikaryon, which are a hallmark feature and the primary cellularphenotype of type A NPD. The presence of distended lysosomes correlateswith the loss of normal cellular function and a progressiveneurodegenerative course that leads to death of the affected individualin early childhood (The Metabolic and Molecular Bases of InheritedDiseases, eds. Scriver et al., McGraw-Hill, New York, 2001, pp.3589-3610). Secondary cellular phenotypes (e.g., additional metabolicabnormalities) are also associated with this disease, notably the highlevel accumulation of cholesterol in the lysosomal compartment.Sphingomyelin has strong affinity for cholesterol, which results in thesequestering of large amounts of cholesterol in the lysosomes of ASMKOmice and human patients (Leventhal et al. (2001) J. Biol. Chem.,276:44976-44983; Slotte (1997) Subcell. Biochem., 28:277-293; and Vianaet al. (1990) J. Med. Genet., 27:499-504.) Other models for other LSDare discussed above and can be used as is appropriate.

Visualization of green fluorescent protein (GFP) expression in mice thathad been treated with AAV4-GFP indicated that GFP was distributedthroughout the ependymal cell layer of the ventricular system. Forexample, GFP was visualized in the anterior lateral ventricles, thelateral ventricles, the third ventricle, and the fourth ventricle (FIG.1). GFP was also visualized in the choroid plexus of the ventricularsystem and the ependymal cell layer of the spinal cord central canal(including the cervical, thoracic, and lumbar regions) (FIG. 2).

Intracerebroventricular injection of recombinant AAV encodingglucocerebrosidase in a neuronopathic mouse model of Gaucher disease

There are animal models of Gaucher disease, which are derived bytargeted disruption of the corresponding mouse gene. For example, thereare models of acute neuronopathic Gaucher disease developed by Enquistet al. 2007 PNAS 104(44): 17483-17488 (incorporated herein by referencein its entirety). These mice exhibit close similarity, in bothpathological findings and clinical manifestations, to human patientswith severe neuronopathic Gaucher disease. An experiment was performedin one of the two models, the K14-lnl/lnl (also referred to herein asthe “K2” mouse). The K2 model is the more severe form of the modelsdescribed in Enquist et al. as the mouse manifests with a more rapidclinical and pathological progression of symptoms.

At one day post-birth, neonatal K2 mice were injected bilaterally usingintracerebroventricular injection into the lateral ventricles with arecombinant adeno-associated virus encoding for human glucocerebrosidase(GC) at a dose of 1.2 e10 genome copies total per brain delivered in avolume of 2 microliters per lateral ventricle. The AAV virus comprisedan AAV5 capsid, AAV2 ITRs, and encoded for human GC (AAV-GC). The K2mice have disruptions in both copies of the GC gene and should have noendogenous expression of GC in their tissues. At one day post-birth, twocontrol groups of littermates of the K2 mice were also injected with thevirus. The first control group contained the “wild-type” mouse group,which have no disruptions in the GC gene and thus normal, endogenous GCexpression. The second control group contained the “heterozygote” mousegroup, which have one disrupted copy of the GC gene and onenon-disrupted copy of the GC gene and thus express some endogenous levelof GC.

The groups of mice were evaluated for total GC enzymatic activity in thebrain and the liver as a representative peripheral organ; reversal ofpathology; weight as surrogate for overall health; extension of lifespanvia a Kaplan-Meier survival curve; and spread of enzyme within thecentral nervous system. To determine mortality in a reliable and humanefashion, an artificial end point is used as defined by when end-stageparalysis in mice was observed.

FIG. 3 is a Kaplan-Meier survival curve demonstrating the survival dataof the AAV-GC treated K2 mice (AAV2/5 GC; represented by triangles) andthe AAV-GC treated wild-type littermate control group (WT; representedby upside-down triangles). Historical survival data from the K2 mice inthe mouse colony has been added to the Kaplan-Meier survival curve(Untreated; represented by boxes). As indicated by the survival data,the AAV-GC treated K2 mice have a significantly longer median survivaltime (median survival=21 days) as compared to the historical mediansurvival time for the untreated K2 mice (median survival=13.5 days). Thedifference between the historical median survival time and the AAV-GCtreated K2 mice median survival time is statistically significant(p<0.0001). The AAV-GC wild-type littermate control group survived untilthe termination of the study. The AAV-GC treated heterozygote littermatecontrol group also survived until the termination of the study (data notshown on figure).

FIG. 4 represents GC activity in the brain and liver of AAV-GC treatedK2 mice (injected as described above) as compared to several controlgroups. The first control group contained the “wild-type” mouse group,which have no disruptions in the GC gene and thus normal, endogenouslevels of GC expression. This group was treated with AAV-GC in the samemanner as the treated K2 mice (labeled AAV5 WT in FIG. 4). The secondcontrol group contained the “heterozygote” mouse group, which have onedisrupted copy of the GC gene and one non-disrupted copy of the GC geneand thus express some level of endogenous GC. These mice were alsotreated with AAV-GC (labeled AAV5 Het in FIG. 4). The final controlgroup also contained the “wild-type” mouse group, but these mice werenot treated with AAV-GC (labeled WT in FIG. 4). As demonstrated in FIG.4, intracerebroventricular injection of AAV encoding for GC resulted inGC enzyme activity in the brain of K2 mice. In these treated K2 mice, GCactivity in the liver was not significantly above background.

FIG. 5 represents the growth of AAV-GC treated K2 mice over time (asmeasured by weight) as compared to the growth over time of AAV-GCtreated wild-type littermates. In this mouse model and regardless ofgenotype, the mouse pups from smaller litters tend to be larger than themouse pups from larger litters. To account for this variation in mousepup size as a function of the litter size, the weight of the K2 mousepups are normalized as a percentage of their own littermates. There isno significant difference between the growth over time of the K2 mice ascompared to the heterozygote littermates (data not shown). In theory,the weight curve for untreated K2 mice should be different than thecurve of the AAV-GC treated K2 mice represented by FIG. 5. Based on thehistorical survival data from the K2 colony, the untreated K2's are deadaround 12-15 days post-birth, which is when the AAV-GC treated K2animals start losing weight.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification. Theembodiments within the specification provide an illustration ofembodiments of the invention and should not be construed to limit thescope of the invention. The skilled artisan readily recognizes that manyother embodiments are encompassed by the invention. All publications,patents, and biological sequences cited in this disclosure areincorporated by reference in their entirety. To the extent the materialincorporated by reference contradicts or is inconsistent with thepresent specification, the present specification will supercede any suchmaterial. The citation of any references herein is not an admission thatsuch references are prior art to the present invention.

Unless otherwise indicated, all numbers expressing quantities ofingredients, cell culture, treatment conditions, and so forth used inthe specification, including claims, are to be understood as beingmodified in all instances by the term “about.” Accordingly, unlessotherwise indicated to the contrary, the numerical parameters areapproximations and may very depending upon the desired properties soughtto be obtained by the present invention. Unless otherwise indicated, theterm “at least” preceding a series of elements is to be understood torefer to every element in the series. Those skilled in the art willrecognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. Such equivalents are intended to beencompassed by the following claims.

REFERENCES

-   1. Lindsay, R. M. Neurotrophic growth factors and neurodegenerative    diseases: therapeutic potential of the neurotrophins and ciliary    neurotrophic factor. Neurobiol Aging 15, 249-51 (1994).-   14. Matsushita, M. Projections from the lowest lumbar and    sacral-caudal segments to the cerebellar nuclei in the rat, studied    by anterograde axonal tracing. J Comp Neurol 404, 21-32 (1999).-   17. Matsushita, M. & Gao, X. Projections from the thoracic cord to    the cerebellar nuclei in the rat, studied by anterograde axonal    tracing. J Comp Neurol 386, 409-21 (1997).-   18. Matsushita, M. & Xiong, G. Projections from the cervical    enlargement to the cerebellar nuclei in the rat, studied by    anterograde axonal tracing. J Comp Neurol 377, 251-61 (1997).-   19. Matsushita, M. & Yaginuma, H. Afferents to the cerebellar nuclei    from the cervical enlargement in the rat, as demonstrated with the    Phaseolus vulgaris leucoagglutinin method. Neurosci Lett 113, 253-9    (1990).-   20. Matsushita, M. & Yaginuma, H. Projections from the central    cervical nucleus to the cerebellar nuclei in the rat, studied by    anterograde axonal tracing. J Comp Neurol 353, 234-46 (1995).-   21. Voogd, J. The cerebellar nuclei and their efferent pathways. in    The rat nervous system (ed. Paxinos, G.) 208-215 (Elsevier Academic    Press, San Diego, 2004).-   22. Dodge, J. C. et al. Gene transfer of human acid sphingomyelinase    corrects neuropathology and motor deficits in a mouse model of    Niemann-Pick type A disease. Proc Natl Acad Sci USA 102, 17822-7    (2005).-   23. Kasarskis, E. J. et al. A controlled trial of recombinant    methionyl human BDNF in ALS: The BDNF Study Group (Phase III).    Neurology 52, 1427-33 (1999).-   26. Chirmule, N. et al. Immune responses to adenovirus and    adeno-associated virus in humans. Gene Ther 6, 1574-83 (1999).-   37. High, K. A. Clinical gene transfer studies for hemophilia B.    Semin Thromb Hemost 30, 257-67 (2004).-   38. Maheshri, N., Koerber, J. T., Kaspar, B. K. & Schaffer, D. V.    Directed evolution of adeno-associated virus yields enhanced gene    delivery vectors. Nat Biotechnol 24, 198-204 (2006).-   40. Confavreux C, Hutchinson M, Hours M M, Cortinovis-Tourniaire P,    Moreau T. Rate of pregnancy-related relapse in multiple sclerosis.    Pregnancy in Multiple Sclerosis Group.-   N Engl J Med. 1998 Jul. 30; 339(5):285-91.-   42. Gensert J M, Goldman J E (1997) Endogenous progenitors    remyelinate demyelinated-   axons in the adult CNS. Neuron 19:197-203.-   43. Gregg C, Shikar V, Larsen P, Mak G, Chojnacki A, Yong V W,    Weiss S. White matter plasticity and enhanced remyelination in the    maternal CNS. J Neurosci. 2007 Feb. 21; 27(8): 1812-23.-   44. Handwerger S, Freemark M. The roles of placental growth hormone    and placental lactogen in the regulation of human fetal growth and    development. J Pediatr Endocrinol Metab. 2000 April; 13(4):343-56.-   45. Lesniak M A, Gorden P, Roth J. Reactivity of non-primate growth    hormones and prolactins with human growth hormone receptors on    cultured human lymphocytes. Clin Endocrinol Metab. 1977 May;    44(5):838-49.-   46. Levison S W, Young G M, Goldman J E (1999) Cycling cells in the    adult rat neocortex preferentially generate oligodendroglia. J    Neurosci Res 57:435-446.-   47. Menn B, Garcia-Verdugo J M, Yaschine C, Gonzalez-Perez O,    Rowitch D, Alvarez-Buylla A (2006) Origin of oligodendrocytes in the    subventricular zone of the adult brain. J Neurosci 26:7907-7918.-   48. Pelton E W, Grindeland R E, Young E, Bass N H. Effects of    immunologically induced growth hormone deficiency on myelinogenesis    in developing rat cerebrum. Neurology. 1977 March; 27(3):282-8.-   49. Peters A, Sethares C (2004) Oligodendrocytes, their progenitors    and other neuroglial cells in the aging primate cerebral cortex.    Cereb Cortex 14:995-1007.-   50. Polito A, Reynolds R (2005) NG2-expressing cells as    oligodendrocyte progenitors in the normal and demyelinated adult    central nervous system. J Anat 207:707-716.-   51. Selenkow H A, Saxena B N, Dana C L. Measurement and    pathophysiologic significance of human placental lactogen. In Pecile    A, Finzi C (eds). The Feto-Placental Unit. Amersterdam, Excerpta    Medica, 1969, p 340.-   52. van Walderveen M A, Tas M W, Barkhof F, Polman C H, Frequin S T,    Hommes O R, Valk J (1994) Magnetic resonance evaluation of disease    activity during pregnancy in multiple sclerosis. Neurology    44:327-329.-   53. Voskuhl R R (2003) Hormone-based therapies in MS. Int MS J    10:60-66.-   54. Zumkeller W. Current topic: the role of growth hormone and    insulin-like growth factors for placental growth and development.    Placenta. 2000 July-August; 21(5-6):451-67.-   55. Belichenko P V, Dickson P I, Passage M, Jungles S, Mobley W C,    Kakkis E D. Penetration, diffusion, and uptake of recombinant human    alpha-1-iduronidase after intraventricular injection into the rat    brain. Mol Genet Metab. 2005; 86(1-2):141-9.-   56. Kakkis E, McEntee M, Vogler C, Le S, Levy B, Belichenko P,    Mobley W, Dickson P, Hanson S, Passage M. Intrathecal enzyme    replacement therapy reduces lysosomal storage in the brain and    meninges of the canine model of MPS I. Mol Genet Metab. 2004;    83(1-2):163-74.-   57. Bembi B, Ciana G, Zanatta M, et al. Cerebrospinal-fluid infusion    of alglucerase in the treatment for acute neuronopathic Gaucher's    disease. Pediatr Res 1995; 38:A425.-   58. Lonser R R, Walbridge S, Murray G J, Aizenberg M R, Vortmeyer A    O, Aerts J M, Brady R O, Oldfield E H. Convection perfusion of    glucocerebrosidase for neuronopathic Gaucher's disease. Ann Neurol.    2005 April; 57(4):542-8.    The disclosure of each reference cited is expressly incorporated    herein.

1. A method to deliver a transgene product which is an enzyme which is deficient in a lysosomal storage disease to a subject, comprising: administering a recombinant neurotrophic viral vector comprising said transgene to at least one ventricle of the brain, whereby said transgene is expressed and the expressed protein product is delivered to affected visceral organs.
 2. The method of claim 1 wherein the viral vector is an AAV vector.
 3. The method of claim 1 wherein the viral vector is AAV4
 4. (canceled)
 5. The method of claim 1 wherein the viral vector is administered by direct injection into a ventricle of the brain.
 6. The method of claim 1 wherein the viral vector is administered by direct injection into a lateral ventricle of the brain.
 7. The method of claim 1 wherein the viral vector is administered by direct injection into the fourth ventricle of the brain. 8-9. (canceled)
 10. A method to treat a lysosomal storage disease in a subject, comprising: administering a recombinant neurotrophic viral vector comprising a therapeutic transgene encoding an enzyme which is deficient in the subject to at least one ventricle of the brain, whereby said transgene is expressed in a therapeutically effective amount.
 11. The method of claim 10 wherein the viral vector is an AAV vector.
 12. The method of claim 10 wherein the viral vector is AAV4
 13. The method of claim 10 wherein the transgene is selected from the group consisting of Aspartylglucosaminidase, alpha-Galactosidase A, Palmitoyl Protein Thioesterase, Tripeptidyl Peptidase, Lysosomal Transmembrane Protein, Multiple gene products, Cysteine transporter, Acid ceramidase, Acid alpha-L-fucosidase, Protective protein/cathepsin A, Iduronate-2-sulfatase, alpha-L-Iduronidase, Galactocerebrosidase, Acid alpha-mannosidase, Acid beta-mannosidase, Arylsulfatase B, Arylsulfatase A, N-Acetylgalactosamine-6-sulfate, Acid beta-galactosidase, N-Acetylglucosamine-1-phosphotransferase, Acid sphingomyelinase, NPC-1, alpha-glucosidase, beta-Hexosaminidase B, Heparan N-sulfatase, alpha-N-Acetylglucosaminidase, Acetyl-CoA:alpha-glucosaminide, N-Acetylglucosamine-6-sulfate, alpha-N-Acetylgalactosaminidase, alpha-N-Acetylgalactosaminidase, alpha-Neuramidase, beta-Glucuronidase, beta-Hexosaminidase A, and Acid Lipase.
 14. The method of claim 10 wherein the viral vector is administered by direct injection into a ventricle of the brain.
 15. The method of claim 10 wherein the viral vector is administered by direct injection into a lateral ventricle of the brain.
 16. The method of claim 10 wherein the viral vector is administered by direct injection into the fourth ventricle of the brain. 17-18. (canceled)
 19. The method of claim 1 wherein said transgene is selected from the group consisting of Aspartylglucosaminidase, alpha-Galactosidase A, Palmitoyl Protein Thioesterase, Tripeptidyl Peptidase, Lysosomal Transmembrane Protein, Multiple gene products, Cysteine transporter, Acid ceramidase, Acid alpha-L-fucosidase, Protective protein/cathepsin A, Iduronate-2-sulfatase, alpha-L-Iduronidase, Galactocerebrosidase, Acid alpha-mannosidase, Acid beta-mannosidase, Arylsulfatase B, Arylsulfatase A, N-Acetylgalactosamine-6-sulfate, Acid beta-galactosidase, N-Acetylglucosamine-1-phosphotransferase, Acid sphingomyelinase, NPC-1, alpha-glucosidase, beta-Hexosaminidase B, Heparan N-sulfatase, alpha-N-Acetylglucosaminidase, Acetyl-CoA:alpha-glucosaminide, N-Acetylglucosamine-6-sulfate, alpha-N-Acetylgalactosaminidase, alpha-N-Acetylgalactosaminidase, alpha-Neuramidase, beta-Glucuronidase, beta-Hexosaminidase A, and Acid Lipase.
 20. The method of claim 1, wherein the subject has a condition selected from the group consisting of Aspartylglucosaminuria, Fabry, Infantile Batten Disease (CNL1), Classic Late Infantile Batten Disease (CNL2), Juvenile Batten Disease (CNL3), Batten, other forms (CNL4-CNL8), Cystinosis, Farber, Fucosidosis, Galactosidosialidosis, G.sub.M1 gangliosidosis, Hunter, Hurler-Scheie, Krabbe, alpha.-Mannosidosis, beta.-Mannosidosis, Maroteaux-Lamy, Metachromatic leukodystrophy, Morquio A, Morquio B, Mucolipidosis Niemann-Pick A, B, Niemann-Pick C, Pompe Acid, Sandhoff, Sanfilippo A, Sanfilippo B, Sanfilippo C, Sanfilippo D, Schindler Disease, Schindler-Kanzaki Sialidosis, Sly, Tay-Sachs, and Wolman Disease. 21-22. (canceled)
 23. The method of claim 1, wherein said subject is a human patient. 24-25. (canceled)
 26. The method of claim 1 wherein the transgene protein product is TAT-modified.
 27. The method of claim 28 wherein the transgene protein product comprises an 11 amino acid motif from the protein transduction domain of HIV TAT protein.
 28. The method of 1, wherein said transgene expresses a therapeutic amount of at least two proteins selected from the group consisting of Aspartylglucosaminidase, alpha-Galactosidase A, Palmitoyl Protein Thioesterase, Tripeptidyl Peptidase, Lysosomal Transmembrane Protein, Multiple gene products, Cysteine transporter, Acid ceramidase, Acid alpha-L-fucosidase, Protective protein/cathepsin A, Iduronate-2-sulfatase, alpha-L-Iduronidase, Galactocerebrosidase, Acid alpha-mannosidase, Acid beta-mannosidase, Arylsulfatase B, Arylsulfatase A, N-Acetylgalactosamine-6-sulfate, Acid beta-galactosidase, N-Acetylglucosamine-1-phosphotransferase, Acid sphingomyelinase, NPC-1, alpha-glucosidase, beta-Hexosaminidase B, Heparan N-sulfatase, alpha-N-Acetylglucosaminidase, Acetyl-CoA:alpha-glucosaminide, N-Acetylglucosamine-6-sulfate, alpha-N-Acetylgalactosaminidase, alpha-N-Acetylgalactosaminidase, alpha-Neuramidase, beta-Glucuronidase, beta-Hexosaminidase A, and Acid Lipase. 29-32. (canceled)
 33. The method of claim 10, wherein the subject has a condition selected from the group consisting of Aspartylglucosaminuria, Fabry, Infantile Batten Disease (CNL1), Classic Late Infantile Batten Disease (CNL2), Juvenile Batten Disease (CNL3), Batten, other forms (CNL4-CNL8), Cystinosis, Farber, Fucosidosis, Galactosidosialidosis, G.sub.M1 gangliosidosis, Hunter, Hurler-Scheie, Krabbe, alpha.-Mannosidosis, beta.-Mannosidosis, Maroteaux-Lamy, Metachromatic leukodystrophy, Morquio A, Morquio B, Mucolipidosis Niemann-Pick A, B, Niemann-Pick C, Pompe Acid, Sandhoff, Sanfilippo A, Sanfilippo B, Sanfilippo C, Sanfilippo D, Schindler Disease, Schindler-Kanzaki Sialidosis, Sly, Tay-Sachs, and Wolman Disease. 